Cisplatin is one of the most widely used chemotherapy agents, often in combination with radiation therapy. Although cells deficient in elements of the Ku/DNA-PK repair complex are extremely sensitive to ionizing radiation, we have found that such cells are markedly resistant to cisplatin. This resistance arises because these cells fail to activate a novel-signaling pathway that depends on the kinase activity of DNA-PK to generate a signal that is passed from cell to cell, inducing death in the recipient. Initial work has demonstrated that this signal is transmitted via gap junctions, since cells defective in gap junction communication are also resistant to cisplatin. These findings suggest that DNA-PK activity and gap junction communication may influence the clinical response to cisplatin in human cancers. We propose to test the hypothesis that the regulation of gap junction expression and function can influence the cellular response to cisplatin. We will investigate links between phosphorylation of connexins (the protein subunits of gap junctions) by selected kinases and cisplatin resistance. We will test the effect of factors that decrease connexin degradation (such as proteasome inhibitors) or enhance gap junction biogenesis (by altering connexin trafficking) on cell killing by cisplatin. To identify factors upstream or downstream of Ku/DNA-PK in response to cisplatin, we will probe candidate damage recognition and signaling factors, using co-immunoprecipitation techniques. We will also use mass spectrometry to identify proteins that show altered binding to Ku80 following cisplatin treatment. Using metabolic labeling and chromatography techniques, we will identify candidate molecules that may transduce the Ku/DNA-PK dependent cytotoxic signal through gap junctions. Finally, we will test the hypothesis that gap junction expression and DNA-PK function can influence cisplatin response in vivo using a series of genetically manipulated tumor models in mice. The long-term goal of this application is to elucidate the molecular mechanisms that mediate this novel cisplatin-response pathway. This work may lead to strategies to sensitize human cancers to cisplatin, providing the basis for new combined approaches to cancer therapy.